Carrying out chemical or biochemical analyses, syntheses or preparations, even at the simplest levels, requires one to perform a large number of separate manipulations on the material components of that analysis, synthesis or preparation, including measuring, aliquoting, transferring, diluting, concentrating, separating, detecting etc.
In developing microfluidic technologies, researchers have sought to miniaturize many of these manipulations and/or to integrate these manipulations within one or a few microscale devices. Many of the above described manipulations easily lend themselves to such miniaturization and integration. For example, the use of these microfluidic technologies has been described in a number of applications, including, e.g., amplification (U.S. Pat. Nos. 5,587,128 and 5,498,392) and separation of nucleic acids (Woolley et al., Proc. Nat""l. Acad. Sci. 91:11348-352 (1994) and hybridization analyses (WO 97/02357 to Anderson).
Despite the application of microfluidic technologies to these manipulations, there are still a number of areas where that application is not so easily made. For example, the performance of large dilutions generally requires the combination of a small volume of the material that is desired to be diluted with a large volume of diluent. By definition, microfluidic systems have extremely small overall volumes, and are typically unable, or less able, to handle the larger volumes required for such dilutions. Further, such large dilutions also typically require the accurate, repeatable dispensing of extremely small volumes of the material to be diluted. However, most microfluidic technology is incapable of accurately dispensing fluid volumes substantially less than a microliter. Although the problems associated with the inability to aliquot extremely small volumes might generally be overcome by performing serial dilutions, such serial dilutions generally require devices with substantially larger volumes, e.g., tens or hundreds of microliters. Specifically, even if one assumes a lower limit of fluid handling of 100 nanoliters, a 1:10 dilution would require a device to handle at least a volume of 1 xcexcl. Further serial dilution steps only increase the required volume.
It would therefor be desirable to provide microfluidic systems which are capable of performing each of the various manipulations required, and which are capable of doing so with a sufficiently small volume whereby, multiple operations can be integrated into a single low volume device or system and performed automatically and with a high degree of precision. Of particular interest would be a microfluidic device or system, as well as methods for using such devices and systems for performing in situ dilution or concentration of a particular material within a microfluidic format. The present invention meets these and many other needs.
The present invention is generally directed to methods, apparatus and systems for performing in situ concentration or dilution of a material in microfluidic devices or systems. In one aspect, the present invention provides microfluidic devices and systems for performing in situ dilution, and particularly in situ serial dilution, of a particular subject material. The devices and/or systems typically comprise a microfluidic device which has at least one main channel disposed therein, where the main channel has at least one microscale cross-sectional dimension. The devices and/or systems also typically comprise at least a first source of the subject material that is to be diluted, in fluid communication with the main channel at a first point along the length of the channel, at least a first source of diluent in fluid communication with the main channel at a second point along the length of the channel, and at least a first reservoir in fluid communication with the main channel at a third point along the length of the channel. The systems of the present invention further comprise a fluid direction for delivering diluent to the main channel to be combined with the subject material to form first diluted material, and for removing at least a portion of the first diluted material from the main channel to the reservoir. Additional diluent sources and reservoirs also may be supplied to further dilute the subject material.
In a closely related aspect, the present invention provides a microfluidic system for continuously diluting a subject material within a microfluidic device. The devices and/or systems typically comprise a microfluidic device which has at least one main channel disposed therein, where the main channel has at least one microscale cross-sectional dimension. The device also typically comprises at least a first source of the subject material in fluid communication with the main channel at a first point along the length of the channel, at least a first source of diluent in fluid communication with the main channel at a second point along the length of the channel, and at least a first reservoir in fluid communication with the main channel at a third point along the length of the channel. The systems of the present invention further comprise a fluid direction system for continuously delivering diluent to the main channel to be combined with the subject material to form first diluted material, and continuously transporting a portion of the first diluted material from the main channel to the reservoir.
The present invention also provides microfluidic systems for in situ concentration of a subject material within a microfluidic device. In this aspect, the system comprises a microfluidic device having a first channel disposed therein, which channel has first, second and third fluid regions disposed therein. The first fluid region typically comprises the subject material and has a first conductivity, whereas the second and third fluid regions are disposed within the first channel on both ends of the first fluid region. The second and third fluid regions have a second conductivity, where the second conductivity is greater than the first conductivity. The system also typically comprises an electroosmotic fluid direction system for transporting the first and second fluid regions along the first channel.
In a further aspect, the present invention provides methods for in situ dilution of a subject material in a microfluidic device. The methods typically comprise combining a first volume of the subject material with a first volume of diluent in a first microscale channel to form a first diluted material. At least a first portion of the first diluted material is then transported out of the first channel. A second volume of diluent from a second diluent source is then delivered to the first microscale channel to combine the second volume of diluent with a second portion of the first diluted material to form a second diluted material.
In a related aspect, the present invention provides a method for in situ dilution of a material in a microfluidic device, which method comprises combining a first volume of said material with a first volume of diluent in a first region of a microfluidic device to form a first diluted material. A portion of the first diluted material is then transported into a second region of the microfluidic device, i.e., a reservoir, where it is combined with a second volume of diluent to form a second diluted material.
In a further aspect, the present invention provides a method for the in situ concentration of a material in a microscale channel. The method comprises introducing a first fluid containing the material into a microscale channel to provide a first fluid region within the channel. The first fluid has a first conductivity, and is bounded by second and third fluid regions, where the second and third fluid regions have a second conductivity which is greater than the first. A voltage gradient is then applied along the length of the microscale channel whereby the first, second and third fluid regions are transported along the length of the microscale channel with a first electroosmotic mobility, and whereby the material in the first fluid has an electrophoretic mobility different from the electroosmotic mobility, resulting in a concentration of the subject material at or near one end of the first fluid region.